1. Field of the Invention
This invention relates to chemical and biochemical analysis using surface-enhanced Raman spectroscopy and, more particularly, it relates to the use of paramagnetic particles to spatially localize an analyte in the presence of a spectral enhancement particle for chemical and biochemical analyses.
2. Description of the Related Art
Raman spectroscopy finds its origins in Planck and Einstein's formulation that light is not only wavelike in nature, but has the dual character of waves and particles. Once scientists began thinking about the concept of light as particles, the possibility of inelastic scattering of these particles became a method of proof of this new theory. In 1923 Compton showed this with inelastic scattering of x-rays from a graphite target. That same year Smekal theoretically predicted that photons should inelastically scatter from molecular transitions. Five years later, in 1928, C. V. Raman and K. S. Krishnan published an article in the journal Nature with experiments that proved Smekal's prediction of inelastic scattering of light. For his discovery, Raman was award the Nobel Prize and the inelastic scattering of visible light from molecular transitions has been named after him.
When light is scattered from a molecule most photons are elastically scattered. The scattered photons have the same energy (frequency) and, therefore, wavelength, as the incident photons. However, a small fraction of light (approximately 1 in 107 photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. The process leading to this inelastic scatter is the termed the Raman effect. Raman scattering can occur with a change in vibrational, rotational or electronic energy of a molecule. The difference in energy between the incident photon and the Raman scattered photon is equal to the energy of a vibration of the scattering molecule. A plot of intensity of scattered light versus energy difference is a Raman spectrum.
The Raman effect arises when a photon is incident on a molecule and interacts with the electric dipole of the molecule. It is a form of electronic (more accurately, vibronic) spectroscopy, although the spectrum contains vibrational frequencies. In classical terms, the interaction can be viewed as a perturbation of the molecule's electric field. In quantum mechanics the scattering is described as an excitation to a virtual state lower in energy than a real electronic transition with nearly coincident de-excitation and a change in vibrational energy. The scattering event occurs in 10−14 seconds or less.
One of the characteristics of inelastic scattering is that the intensity of the scattering scales to the fourth power of the energy. This means that Compton's experiments with X-rays with a wavelength of 0.7 nm and the observation of Raman scattering with visible light at 500 nm will differ by 11 orders of magnitude. Raman was able to observe the weak Raman effect by using the most intense light source available at the time, the sun. He focused a large telescope on the sun and placed a green filter in the intense beams of sunlight. When he used a yellow filter to observe this beam of green light passing through a solution of chloroform, he could see a weak yellow light. The origin of the yellow light was the Raman effect. A small amount of the green light from the sun had inelastically scattered from the chloroform molecules and shifted its energy so that the photons fell within the yellow part of the spectrum.
The Raman effect is observed as a shift in energy of a photon and the shift can be related to a vibrational state of the sample. To observe the shift all of the photons need to be within a very narrow band of energies, otherwise, it is difficult to distinguish the shifted photons from the source photons. Raman spectroscopy is conventionally performed with a green, red or near-infrared laser. The laser produces quasi-monochromatic light that forms a very narrow band of wavelengths. The laser also produces this light in a small concentrated beam that is very intense. The wavelength of the laser is below the first electronic transitions of most molecules, as assumed by scattering theory. However, if the wavelength of the exciting laser is within the electronic spectrum of a molecule, the intensity of some Raman-active vibrations increases by a factor of 102-104. This resonance enhancement or resonance Raman effect can be quite useful.
The Raman scattering from a compound (or ion) adsorbed on or even within a few Angstroms of a structured noble metal surface can be 103-1015 times greater than in solution. This surface-enhanced Raman scattering (SERS) is strongest on silver and gold, but is observable on copper as well. At practical excitation wavelengths, significant enhancement on other metals has not been observed.
Surface-enhanced Raman scattering arises from two mechanisms. The first is an enhanced electromagnetic field produced at the surface of the metal. When the wavelength of the incident light is close to the plasma wavelength of the metal, conduction electrons in the metal surface are excited into an extended surface electronic excited state called a surface plasmon resonance. Molecules adsorbed on or in close proximity to the surface experience an exceptionally large electromagnetic field. Vibrational modes normal to the surface are most strongly enhanced. The second mode of enhancement is by the formation of a charge-transfer complex between the surface and analyte molecule. The electronic transitions of many charge transfer complexes are in the visible spectrum leading to resonance enhancement.
The intensity of the surface plasmon resonance is dependent on many factors including the wavelength of the incident light and the morphology of the metal surface. The wavelength should match the plasma wavelength of the metal. This is about 382 nm for a 5 μm silver particle, but can be much higher for larger ellipsoidal silver particles. The plasma wavelength is to the red of 550 nm (i.e., wavelengths greater than 550 nm) for copper and gold, the other two metals which show SERS at wavelengths in the 350-1000 nm region. The best morphology for surface plasmon resonance excitation is a small (<100 nm) particle or an atomically rough surface.
Molecules with lone pair electrons or pi clouds show the strongest SERS. The effect was first discovered with pyridine. Other aromatic nitrogen or oxygen containing compounds, such as aromatic amines or phenols, are strongly SERS active. The effect can also been seen with other electron-rich functionalities such as carboxylic acids.
SERS is used to study monolayers of materials adsorbed on metals, including electrodes. Many formats other than electrodes can be used. The most popular include colloids, metal films on dielectric substrates, and arrays of metal particles bound to metal or dielectric colloids through short linkages. Although SERS allows easy observation of Raman spectra from solution concentrations in the micromolar (1×106) range, slow adsorption kinetics and competitive adsorption limit its application in analytical chemistry.
In 1991 Carron, et al., demonstrated that SERS surfaces could be used to detect trace amounts of materials. (See Ultrasensitive Detection of Metal Ions with Surface Enhanced Raman Spectroscopy. K. Carron, K. Mullen, H. Angersbach, and M. Lanouette, Appl. Spectrosc., 45:420 (1991).) This introduces the concept of a coating on a SERS particle that has an affinity for an analyte. Carron defined three types of coatings: passive, active, and reactive. A passive coating creates an affinity for an analyte and the analyte is detected from the analyte's Raman spectrum. Passive coatings attract the analyte in a reversible fashion. An active coating has an affinity for an analyte and the analyte is detected through a change in the spectrum of the coating. Active coatings also bind the analyte reversibly. A reactive coating reacts with the analyte and produces a new molecular coating that has incorporated the analyte into its chemical structure. In this case, the analyte is detected by a change in the Raman spectrum of the coating. Reactive coatings bind the analyte irreversibly.
In 1993 Tarcha, et al., U.S. Pat. No. 5,266,498, the applicants describe a similar approach using an antibody on a SERS surface to act as an affinity. This reference discloses applying an antibody to a SERS surface. The antibody on the SERS surface, in turn, binds to an analyte. After the analyte is bound to the antibody on the surface of a SERS particle, a second antibody having a resonance Raman active dye conjugated to it is added. The second antibody also has an affinity for the analyte and similarly binds to the analyte. Traditionally this is known as a sandwich assay. When the dye bound to the second antibody is near the SERS active surface, a Raman spectral signal of the dye is observed.
The information included in this Background section of the specification, including any references cited herein and any description or discussion thereof, is included for technical reference purposes only and is not to be regarded subject matter by which the scope of the invention is to be bound.